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Production of biologically active recombinant bovine gonadotropins using the baculovirus expression system
Abstract
Biologically active recombinant bovine gonadotropins were produced using the baculovirus expression system. Bovine gonadotropins subunit cDNAs for alpha, follicle stimulating hormone (FSH) $\beta$ and luteinizing hormone (LH) $\beta$ were subcloned into the baculovirus transfer plasmid pFastBac1 for generation of recombinant baculoviruses as bacmids (Bac-to-Bac Expression System, Life Technologies, Gaithersburg, MD). Recombinant baculoviruses were amplified and titered using Sf9 cells while proteins were expressed in High Five cells. Monomeric alpha and FSH$\beta$ were produced at rates of 6-8 $\mu$g/million cells under optimal expression conditions. Co-infection of alpha and FSH$\beta$ baculoviruses yielded a production rate of 1-2.5 $\mu$g/million cells for heterodimeric FSH. Recombinant bovine FSH bound to human receptors expressed on CHO cells with a potency approximately 7-fold higher and induced signal transduction in a Y1 cell bioassay with a potency approximately 2.5-fold higher, than pituitary derived bovine FSH. Only a small percentage of monomeric LH$\beta$ appeared in the medium when expressed under optimal conditions. Recombinant LH$\beta$ released in the medium generated a curve parallel to the standard in the radioimmunoassay, while the dose-response curve for LH$\beta$ in cell extracts was non-monotonic suggesting intracellular retention and degradation. Co-expression of recombinant alpha and LH$\beta$ baculoviruses resulted in much higher $(>$10 fold) secretion rates suggesting that subunit assembly stabilizes recombinant LH$\beta.$ Thus, a significant percentage of monomeric bovine LH$\beta$ appears to be retained in High Five insect cells and degraded unless rescued by combination with an alpha subunit. Heterodimeric LH expressed in insect cells was bioactive as judged by its ability to stimulate progesterone secretion in cultured MA-10 Leydig tumor cells. A bovine LH$\beta$ analog with the equine LH/CG$\beta$ carboxyl terminal peptide (CTP) domain was constructed creating a chorionic gonadotropin (CG)-like molecule to determine if addition of a CTP domain could enhance secretion. The CTP fragment of an equine LH/CG$\beta$ cDNA was subcloned into pFastBac1.bLH$\beta$ generating a DNA sequence that encodes a bovine LH$\beta\sp{1-110}$-equine LW/CG$\beta\sp{111-149}$ fusion protein (bLH$\beta$eqCTP). Under optimal infection conditions, the secretion rate of bovine LH$\beta$eqCTP was 0.4 $\mu$g/million cells. Like monomeric bovine LH$\beta,$ bovine LH$\beta$eqCTP appeared to be retained inside the cells unless rescued by combination with an alpha subunit. The bovine LH$\beta$eqCTP fusion protein was able to bind to LH receptors and induce signal transduction in cultured MA-10 Leydig tumor cells. Thus, adding a CTP sequence to bovine LH$\beta$ does not markedly enhance secretion of the monomeric form. Therefore, the determinants of LH$\beta$ which encode for intracellular retention are likely upstream from the carboxy-terminal.
Subject Area
Anatomy & physiology|Animals|Biochemistry
Recommended Citation
Chadha, Prabhjit Kaur, "Production of biologically active recombinant bovine gonadotropins using the baculovirus expression system" (1997). ETD collection for University of Nebraska-Lincoln. AAI9805499.
https://digitalcommons.unl.edu/dissertations/AAI9805499